The long term objective of the proposed project is to understand the transcriptional regulation of the intracellular form of interleukin-1 receptor antagonist (iclL-1ra) and the role it plays in the modulation of IL-1 mediated inflammatory diseases such as rheumatoid arthritis. iclL-1ra is produced constitutively in epithelial cells and recent evidence suggests that it is also inducible in other types of cells involved in IL- 1 mediated disease, namely monocytes/macrophages and fibroblasts. The specific aims of this proposal are: to define the cell-type specificity of iclL-1ra promoter expression in cell types involved in IL-1-mediated inflammatory disease (keratinocytes, fibroblasts and monocyte/macrophages); to further characterize the patterns of inducibility of the iclL-1ra gene in these cell types; to identify the specific cis-acting DNA elements and unique transcriptional factors responsible for both basal transcription in epithelial cells and inducibility in epithelial and non-epithelial cells. In preliminary work a 4555 bp sequence 5' of the transcriptional start site of the human iclL-1ra gene has been cloned into a luciferase expression vector. This construct (plC4555Luc) has been transfected into keratinocyte and macrophage cell lines revealing a similar pattern of transcriptional activation as that of endogenous iclL-1ra protein expression. To achieve the specific aims the inducibility of the iclL-1ra gene will be further characterized in monocytes/macrophages, fibroblasts and keratinocytes using ELISA, Western and RT-PCR techniques. Of particular interest is a unique LPS response element and transcriptional factors possibly present in monocyte/macrophages and fibroblasts which may regulate transcription of iclL-1ra but not sIL-1ra. Gene transfer experiments will examine the tissue-specific basal and induced expression of the iclL-1ra gene. Gene transfer experiments with 5' deletion mutants, DNase 1 protection assays and electrophoretic mobility shift assays will be used to localize specific cis-acting DNA regulatory elements and identify the binding factors necessary for basal and inducible gene expression. Heterologous promoter constructs will examine the function of promoter elements outside their normal context. The environment is uniquely suited to this project because the candidate will be working closely with Dr. M. Smith who has studied the transcriptional regulation of slL-1ra, and there is an active program of research in transcriptional regulation at the institution. The laboratory in which the candidate will be working is involved in many aspects of IL-1 and IL-1ra biology and has experience with the techniques which will be utilized for the proposed project.